The tryptic peptides of rabbit muscle triose phosphate isomerase

1. The peptides obtained by tryptic digestion of S-[(14)C]carboxymethylated rabbit muscle triose phosphate isomerase have been studied. 2. The first step in the fractionation of the tryptic digest was gel filtration on coupled columns of Sephadex G-25 and G-50. Further fractionation was carried out by paper electrophoresis and paper chromatography. 3. The digest contained 26 peptides and three free amino acids. The sizes of the peptides ranged from two to 29 residues. 4. The sequences of the peptides have been determined. 5. The length of the polypeptide chains is about 250 amino acid residues. 6. The variant sequences encountered were due to partial deamidation; this may be one of the reasons for multiple forms of the enzyme. 7. The chicken and rabbit enzymes are compared. 8. Detailed evidence for the sequences of the tryptic peptides has been deposited as Supplementary Publication SUP 50024 at the British Library, Lending Division (formerly the National Lending Library for Science and Technology), Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms given in Biochem. J. (1973) 131, 5.     

Studies on the subunit structure and amino acid sequence of triose phosphate isomerase from chicken breast muscle

1. Triose phosphate isomerase was prepared by chromatography on DEAE-cellulose of an (NH(4))(2)SO(4) fraction of an extract of homogenized chicken breast muscle. The product is homogeneous on gel electrophoresis and is suitable for growing crystals for X-ray work. The specific activity is 10000 units/mg and the value for E(0.1%) (280) is 1.20. 2. Comparison between the sum of the amino acid compositions of the tryptic peptides of the protein and the amino acid composition obtained on total hydrolysis of the protein indicates that the relative subunit mass is about 27000. 3. These data, together with the results of the examination of the amino acid compositions of a number of minor peptides, the number of peptides in the tryptic digest and the complete amino acid sequences of the tryptic peptides (the determination of which is described here), give no indication that the subunits are dissimilar. 4. A tentative amino acid sequence is presented for the protein, in which the ordering of the tryptic peptides is derived by homology with the sequence of the rabbit muscle enzyme (Corran & Waley, 1973). 5. An appendix describes the use that was made of mass spectrometry in the determination of some of the sequences. Mass-spectrometric data have been obtained for 35 residues, that is about 15% of the total sequence of the protein. 6. An extended version of the present paper has been deposited as Supplementary Publication SUP 50025 at the British Library, Lending Division (formerly the National Lending Library for Science and Technology), Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies may be obtained on the terms given in Biochem. J. (1973) 131, 5.     

Amino acid sequences around the cysteine residues of rabbit muscle triose phosphate isomerase

1. The nature of the subunits in rabbit muscle triose phosphate isomerase has been investigated. 2. Amino acid analyses show that there are five cysteine residues and two methionine residues/subunit. 3. The amino acid sequences around the cysteine residues have been determined; these account for about 75 residues. 4. Cleavage at the methionine residues with cyanogen bromide gave three fragments. 5. These results show that the subunits correspond to polypeptide chains, containing about 230 amino acid residues. The chains in triose phosphate isomerase seem to be shorter than those of other glycolytic enzymes.     

The active centre of rabbit muscle triose phosphate isomerase. The site that is labelled by glycidol phosphate

1. Glycidol (2,3-epoxypropanol) phosphate is a specific irreversible inhibitor of rabbit muscle triose phosphate isomerase (EC 5.3.1.1); the site of attachment has now been studied. 2. The labelled enzyme was digested with pepsin and a modified peptide isolated. The sequence of the peptide is: Ala-Tyr-Glu-Pro-Val-Trp. 3. It is the glutamic acid residue in this peptide that is labelled: the peptide is thus a gamma-glutamyl ester derived from glycerol phosphoric acid. The same site is labelled by a mixture of glycidol and inorganic phosphate. 4. Kinetic and stereochemical features of these reactions are discussed.     

Equilibrium unfolding mechanism of chicken muscle triose phosphate isomerase

Triose phosphate isomerase (TIM) was prepared and purified from chicken breast muscle. The equilibrium unfolding of TIM by urea was investigated by following the changes of intrinsic fluorescence and circular dichroism spectroscopy, and the equilibrium thermal unfolding by differential scanning calorimetry (DSC). Results show that the unfolding of TIM in urea is highly cooperative and no folding intermediate was detected in the experimental conditions used. The thermodynamic parameters of TIM during its urea induced unfolding were calculated as DeltaG degrees =3.54 kcal.mol(-1), and m(G) = 0.67 kcal.mol(-1)M(-1), which just reflect the unfolding of dissociated folded monomer to fully unfolded monomer transition, while the dissociation energy of folded dimer to folded monomer is probe silence. DSC results indicate that TIM unfolding follows an irreversible two-state step with a slow aggregation process. The cooperative unfolding ratio, DeltaH(cal)/DeltaH(vH), was measured close to 2, indicating that the two subunits of chicken muscle TIM unfold independently. The van't Hoff enthalpy, DeltaH(vH), was estimated as about 200 kcal.mol(-1). These results support the unfolding mechanism with a folded monomer formation before its tertiary structure and secondary structure unfolding.     

Refolding of triose phosphate isomerase

The refolding and reactivation of the glycolytic enzyme triose phosphate isomerase (EC 5.3.1.1) has been studied. The enzyme, which is a dimer, is disaggregated and unfolded in solutions of guanidinium chloride. Unfolding, followed by changes in E(233), took place quite rapidly in 3m-guanidinium chloride (i.e. with a half-life of about 1 min). Refolding also took place rapidly when the solution was diluted about tenfold; two first-order processes could be resolved. Regain of enzymic activity was followed by diluting the solution of the denatured enzyme in guanidinium chloride into assay mixture. The half-life (i.e. the time when the activity was half the final activity) depended markedly on the concentration of protein at low concentrations (about 100ng/ml), but at higher concentrations the half-life became independent of concentration. Thus at low concentrations dimerization was a rate-determining step and this is taken to indicate that the monomers showed little or no activity under these conditions. The rate of regain of enzymic activity was the same as the rate of the slower process of refolding, which was detected spectroscopically. The native enzyme was resistant to proteolysis; high concentrations of subtilisin prevented regain of activity, but at lower concentrations refolding competed with proteolysis.     

The active centre of triose phosphate isomerase

1. Cystamine (2,2'-diaminodiethyl disulphide) caused an unmasking of mitochondrial adenosine triphosphatase and a leakage of Mg(2+) from the mitochondria, and decreased the stimulation of adenosine triphosphatase by 2,4-dinitrophenol. When Mg(2+) was added, cystamine potentiated the activation of adenosine triphosphatase by 2,4-dinitrophenol. 2. Cystamine was without effect on the adenosine triphosphatase of disrupted mitochondria. 3. Cystamine was moderately potent as an uncoupling agent and as an inhibitor of the [(32)P]P(i)-ATP exchange reaction. 4. Cysteamine (2-aminoethanethiol) was without the above effects, when special precautions were taken to counteract its autoxidation. 5. The effects of cystamine should probably be ascribed to its disulphide group, since the diamine cadaverine protected slightly against the loss of Mg(2+) and the decrease of 2,4-dinitrophenol-stimulated adenosine-triphosphatase activity caused by aging of the mitochondria. It is suggested that cystamine acts by a breakdown of mitochondrial permeability barriers.     

Specificity and kinetics of triose phosphate isomerase from chicken muscle

The isolation of crystalline triose phosphate isomerase from chicken breast muscle is described. The values of k(cat.) and K(m) for the reaction in each direction were determined from experiments over wide substrate-concentration ranges, and the reactions were shown to obey simple Michaelis-Menten kinetics. With d-glyceraldehyde 3-phosphate as substrate, k(cat.) is 2.56x10(5)min(-1) and K(m) is 0.47mm; with dihydroxyacetone phosphate as substrate, k(cat.) is 2.59x10(4)min(-1) and K(m) is 0.97mm. The enzyme-catalysed exchange of the methyl hydrogen atoms of the ;virtual substrate' monohydroxyacetone phosphate with solvent (2)H(2)O or (3)H(2)O was shown. This exchange is about 10(4)-fold slower than the corresponding exchange of the C-3 hydrogen of dihydroxyacetone phosphate. The other deoxy substrate, 3-hydroxypropionaldehyde phosphate, was synthesized, but is too unstable in aqueous solution for analogous proton-exchange reactions to be studied.     

Pea seed triose phosphate isomerase

Triose phosphate isomerase was purified ca 250-fold from pea seed extracts. The k_m for D-glyceraldehyde-3-P was 0.44 mM and for dihydroxyacetone-P, 0.88 mM. P-enolpyruvate, 2-P-glycerate, 3-P-glycerate and 2-P-glycolate were strongly inhibitory. Pi and arsenate also inhibited pea seed triose phosphate isomerase.     

The amino acid sequence of rabbit skeletal muscle glycogenin

The amino acid sequence of glycogenin from rabbit skeletal muscle has been determined. The N-acetylated protein consists of 332 amino acids and has a molecular mass of 37278 Da. The novel tyrosyl-glucose linkage between glycogenin and glycogen [Smythe, C., Caudwell, F. B., Ferguson, M. & Cohen, P. (1988) EMBO J. 7, 2681-2686] is shown to occur at a single site, tyrosine-194. Although glycogenin is a UDP-Glc utilising glucosyltransferase that self-glucosylates [Pitcher, J., Smythe, C. & Cohen, P. (1988) Eur. J. Biochem. 176, 391-395], following addition by an unknown enzyme of the first glucose to tyrosine-194, it is not homologous to either human glycogen synthase or other UDP-Glc-requiring enzymes.     

The complete amino acid sequence of rabbit muscle phosphoglucomutase

The complete amino acid sequence of rabbit muscle phosphoglucomutase has been determined by isolating the 11 peptide fragments produced by the cyanogen bromide cleavage reaction and subjecting these to automated sequencing procedures. Products produced by treatment of some of these fragments with hydroxylamine, iodosobenzoic acid, mild acid, cyanogen bromide in formic and heptafluorobutyric acids, Staphylococcus aureus V8 protease, and trypsin (with or without blocking at lysine residues) were used to complete the sequence for each of the cyanogen bromide fragments. The cyanogen bromide fragments were ordered by isolating the four tryptic peptides produced by a limited tryptic digest of the native enzyme in the presence of its substrates and its bivalent metal ion activator, Mg2+, degrading these by means of trypsin, after blocking digestion at lysine residues, and isolating and identifying all fragments thus produced that contained 10 or more residues. The 561-residue sequence thus obtained is one of the longest that has been determined by chemical means. There is excellent agreement between this sequence and published compositions after appropriate normalization. The absorbance of the enzyme is about 7.0 at 278 nm for a 1% solution; this value is 9% lower than that previously used.